Abstract Venezuelan Equine Encephalitis Virus (VEEV) is an alphavirus that is classified as a category B select agent and an emerging infectious agent. There are no FDA approved therapeutics or vaccines currently available for treatment of VEEV infection. Our research over the past two years have uncovered that host kinases play an important role in the establishment of a productive infection in VEEV infected cells. Inhibition of host kinase activity by small molecule inhibitors decreases viral load and improves host survival in vitro and in vivo. We have identified that the viral protein nsP3 interacted with a host kinase (IKK???which prompted us to consider the phosphorylation status of nsP3 in infected cells. Our analysis revealed multiple phosphorylated residues on nsP3, many of which were susceptible to our small molecule inhibitor treatment. We have also determined that in addition to IKK??? nsP3 interacted with DDX-1 and DDX-3 in infected cells. An unmet need is a better understanding of the phosphorylation events that occur on nsP3 and the impact of those events on the protein:protein interactions involving nsP3 and host proteins. Understanding the importance of nsP3 phosphorylation to viral multiplication will pave the way for effective therapeutics and rationally designed vaccines. Our hypothesis is that nsP3 will be phosphorylated on multiple residues in infected cells and at least some of these phosphorylation events will be critical to viral multiplication. In addition, the phosphorylation status of nsP3 will impact its ability to interact with the host proteins in the infected cell. As a consequence, when target phosphorylation sites are mutated in the virus, the virulence of the virus will be reduced in vivo. We will test our hypothesis with the following specific aims: Aim 1. Phosphorylation of nsP3 in infected cells. Aim 2. Interaction of nsP3 with host proteins in infected cells.